a. Field of the Invention
The present invention relates generally to biosensors, and more particularly to fluorescence-based biosensors that incorporate polymerase chain reaction (PCR) technology.
b. Description of the Prior Art
The detection of pathogens in air, water, and food, as well as in biological samples, has been of great interest. Tests that detect the presence of and identify a pathogen are also useful in detecting and countering the use of biological weapons. Most commercial systems for the detection of pathogens rely on antibody-based systems. These systems, while useful, have limited sensitivity and are more vulnerable to neutralization by changes in the surface antigenicity of the organism than the lesser used nucleic acid-based detection systems. Probes for nucleic acids can also detect the presence of foreign genes that have been introduced into a different host.
DNA-based detection systems detect a specific pathogen by detecting DNA fragments unique to that pathogen. Additionally, the detection of DNA can be useful in forensic medicine.
Kemp et al, Proc. Natl. Acad Sci. USA, 86 pp 2423-2427 (1989) (incorporated by reference herein), describe a colorimetric scheme for the detection of DNA from a specific organism. DNA from the target organism is amplified in the sample by a process such as PCR using an appropriate homologous primer pair for the DNA sequence to be amplified. Then, the amplified target DNA is further amplified, this time using modified new primers to produce amplified blunt-ended DNA having affinity binding ligands at both ends. The blunt-ended DNA is then affinity bound at one end to a substrate, such as a microtiter dish precoated with an affinity reagent, and at the other end to an enzyme, for example by biotin-avidin bonding. A chromogenic substrate is then added to the dish and allowed to react. The absorbance is then read, for example in a microtiter plate reader. This enzyme-linked assay, while useful, requires considerable work and special equipment. Additionally, the format of the assay makes the detection of multiple different microorganisms difficult.
Fluorescence-based biosensors, relying on antibodies for detection, were known before the present invention, as described in Bhatia et al "Fiber Optic-based Immunosensors: A Progress Report", SPIE Vol. 1054 Fluorescence Detection III (1989), the entirety of which is incorporated herein by reference. Nevertheless, to date, no one has developed a fluorescent sensor-based system for DNA detection using PCR.